Method for identifying mycobacterial species by comparative sequence analysis of rpoB gene

ABSTRACT

The present invention relates to a method for detecting and identifying mycobacterial species which comprises steps of amplifying 342 bp of rpoB gene fragments from clinically isolated mycobacterial using mycobacterial rpoB-specific PCR primers; sequencing 306 bp regions of the amplified 342 bp of rpoB gene fragments except the primer regions; and, inferring a phylogenetic tree with reference species. In accordance with the present invention, it was found that rpoB sequences from 44 mycobacterial species provide a basis for systematic phylogenetic relationship which can be used to identify clinically isolated mycobacteria that are pathogenic or potentially so. Accordingly, the amplification of rpoB DNA followed by automated sequencing and the analysis of phylogenetic relationships with the reference species can be used efficiently to detect and identify clinical isolates of mycobacteria which have not been identified by the conventional methods. In particular, this approach is useful for slowly growing, fastidious or uncultivable mycobacteria. Furthermore, in the case of  M. tuberculosis , rifampin susceptibility can be simultaneously determined. Thus, the PCR- mediated comparative sequence analysis of rpoB DNA of the invention can be regarded as a reliable and rapid method for the diagnosis of mycobacterial infection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for diagnosis of mycobacterial infection by comparative sequence analysis of rpoB gene coding for β-subunit of RNA polymerase, more specifically, to a method for detecting and identifying mycobacterial species which comprises steps of amplifying 342 bp of rpoB gene fragments from clinically isolated mycobacteria using mycobacterial rpoB-specific PCR primers; sequencing 306 bp regions of the amplified 342 bp of rpoB gene fragments except the primer region; and, inferring a phylogenetic tree with reference species.

2. Description of the Prior Art

The genus Mycobacterium covers a wide range of organisms including obligate parasites causing serious human and animal diseases such as tuberculosis, bovine tuberculosis and leprosy, opportunistic pathogens, and saprophytic species found in natural environment (see: Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, Manual of Clinical Microbiology, 6th Ed. ASM Press, Washington, D.C., Frederick S. N., B. Metchock, pp. 400-437(1995)).

Recently, in line with rapid increase of AIDS patients worldwide, infections with nontuberculous mycobacteria or mycobacteria other than Mycobacterium tuberculosis (hereinafter, referred to as “MOTT”) or Mycobacterium leprae go on increasing (see: Barnes, P., A. B. Bloch, P. T. Davidson and D. E. Snider, Jr., Tuberculosis in Patients with Immunodeficiency Virus Infection, New Engl. J. Med., 324:1644-1650(1991)).

In general, mycobacteria have been largely classified into four groups depending on growth rate and pigmentation of colonies(see: Runyon, E. H., Identification of Mycobacterial Pathogens Utilizing Colony Characteristics, Am. J. Clin. Pathol., 54:578-586(1970)), and numerical taxonomic analysis(see: Sneath, P. H. A. and R. R. Sokal, Numerical Taxonomy, W.H. Freeman & Co., San Francisco (1973)), immunological techniques (see: Wayne, L. G., R. C. Good, A. Tsang, R. Buttler, D. Dawson, D. Groothuis, W. Gross, J. Hawkins, J. Kilburn, M. Kubin, K. H. Schroder, V. A. Silcox, M.-F. Thorel, C. Woodley and M. A. Yakrus, Serovar Determination and Molecular Taxonomic Correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: A Cooperative Study of the international Working Group on Mycobacterial Taxonomy, Int. J. Syst. Bacteriol., 43(3):482-489(1993)), comparison of cell wall composition, DNA-DNA homology, and analysis employing restriction endonucleases (see: Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Botter and T. Bodmer, Rapid Identification of Mycobacteria to the Species Level by Polymerase Chain Reaction and Restriction Enzyme Analysis, J. Clin. Microbiol., 31(2):175-178(1993)) have been used to classify mycobacterial species more definitely.

However, the conventional methods for identifying mycobacterial species have revealed disadvantages that they are laborious and complex, and require so long time. Naturally, alternative methods for identification employing a gene as a marker, e.g., genus-specific or species-specific PCR primers or nucleic acid probe against a specific gene have been used in the art.

Under the circumstances, mycobacterial phylogenetic analysis to provide a criterion of differentiation and identification of mycobacterial species has been performed based on the sequences of 16S rRNA or its coding gene (16S rDNA), which provided a fact that 16S rRNA-based phylogenetic analysis permits to define mycobacterial phylogenetic relationships well (see: Stahl, D. A. and J. W. Urbance, The Division between Fast and Slow-growing Species Corresponds to Natural Relationships among the Mycobacteria, J. Bacteriol., 172:116-124(1990); Rogall, T., T. Flohr and E. C. Bottger, Differentiation of Mycobacterium Species by Direct Sequencing of Amplified DNA, J. Gen. Microbiol., 136(Pt9):1915-1920(1990); Rogall, T., J. Wolters, T. Flohr and E. C. Bottger, Towards a Phylogeny and Definition of Species at the Molecular Level within the Genus Mycobacterium, Int. J. Syst. Bacteriol., 40:323-330(1990)).

However, 16S rRNA-based phylogenetic analysis has a shortcoming that clear definition of species boundaries is often difficult (for example, in the case of slow-growing mycobacteria) (see: Fox, G. E., J. D. Wisotzkey and P. J. Jurtshumk, How Close IS Close: 16S rRNA Sequence Identitiy May Not Be Sufficeint to Guarantee Species Identity, Int. J. Syst. Bacteriol., 42:166-170(1992)).

Thus, a dnaJ gene coding for a stress protein has been suggested as a promising alternative (see: Takewaki, S. K. Okuzumi, H. Ishiko, K. Nakahara, A. Ohkubo and R. Nagai, Genus-specific Polymerase Chain Reaction for the Mycobacterial dnaj Gene and Species-specific Oligonucleotide Probes, J. Clin. Microbiol., 31:446-450(1993); Takewaki, S., K. Okuzumi, I. Manabe, M. Tanimura, K. Miyamura, K. Nakahara, Y. Yazaki, A. Ohkubo and R. Nagai, Nucleotide Sequence Comparison of the Mycobacterial dnaJ Gene and PCR-restriction Fragment Length Polymorphism Analysis for Identification of Mycobacterial Species, Int. J. Syst. Bacteriol., 44:159-166(1994)). However, it was found that dnaJ-based phylogenetic analysis has several problems unsuitable for differentiation of rapid-growing mycobacteria.

The said nucleic acid probes to employ 16S rRNA gene as a marker are clear criteria for defining 5 kinds of mycobacterial species, e.g., M. tuberculosis, M. bovis, MAC, M. kansasii and M. gordonae, and they are commercially available in the art (AccuProbe: Gen-Probe, San Diego, Calif., USA) (see: Nolte, F. S. and Beverly Metchock, Ch. 31. Mycobacterium in Manual of Clinical Microbiology, pp. 400-437(1995)).

Also, IS6110 insertion element which exists in TB complex (M. tuberculosis, M. africanum and M. bovis) in multiple copies, has been employed as a marker in a PCR detection method. However, the result obtained through the said method maybe false negative, since Mycobacterium tuberculosis free of the insertion element has been reported (see: Yuen, L. K., B. C. Ross, K. M. Jackson and B. Dwyer, Characterization of Mycobacterium tuberculosis Strains from Vietnamese Patients by Southern Blot Hybridization, J. Clin. Microbiol., 31:1615-1618(1993)). Primers to amplify the said gene are commercially available now (TB-CR, TB Detection kit, Bioneer Co., Korea), though they play a limited role of detecting mycobacterial species, and can not practically be applied in identifying mycobacteria as well as detecting existence of mycobacteria.

When the afore-mentioned 5 kinds of probes are used, typical mycobacterial species causing human diseases can be identified. However, the probes have a shortcoming of cross-hybridization with newly described species (see: Buttler, W. R., S. P. O'connor, M. A. Yakrus and W. M. Gross, Cross-reactivity of Genetic Probe for Detection of Mycobacterium tuberculosis with Newly Described Species Mycobacterium celatum, J. Clin. Microbiol., 32(2):536-538(1994)). Also, infections with MOTT other than Mycobacterium tuberculosis increase, MAIS (Mycobacterium avium-intracellulare-scrofulaceum) complex among MOTT infects human frequently, and infections with new species have been reported continuously. Accordingly, there are strong reasons for clear definition of species causing diseases to prevent and control the diseases, and development of a novel nucleic acid probe showing species-specific genetic difference is strongly required in the art.

Under the circumstances, the present inventors have investigated whether a rpoB gene coding for β-subunit of RNA polymerase is useful as a criterion for mycobacterial phylogenetic analysis, based on the following reports:

RNA polymerase gene, besides the said criteria for phylogenetic analysis, can be used as an alternative of 16S rRNA gene, since RNA polymerase has subunits of rpoA, rpoB, rpoc and rpoD which are highly conserved throughout procaryotes (see: Lill, U. I., E. M. Behrendt and G. R. Hartmann, Eur. J. Biochem., 52:411-420(1975)).

Among the subunits, the rpoB gene coding for β-subunit of RNA polymerase is related to rifampin resistance in Escherichia coli (see: Jin, D. and C. A. Gross, Mapping and Sequencing of Mutations in the Escherichia coli rpoB Gene that Lead to Rifampicin Resistance, J. Mol. Biol., 202:45-58(1988)), Mycobacterium tuberculosis (see: Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer and T. Bodmer, Detection of Rifampin-resistance Mutations in Mycobacterium tuberculosis, Lancet, 341:647-650(1993)), Mycobacterium leprae (see: Honore, N. T., Bergh, S., Chanteau, S., Doucet-Populaire, F., Eiglmeier, K., Garnier, T., Georges, C., Launois, P., Limpaiboon, T., Newton, S., Niang, K., Del Portillo, P., Ramesh, G. R., Reddi, P., Ridel, P. R., Sittisombut, N., Wu-Hunter, S. and Cole, S. T., Nucleotide Sequence of the First Cosmid from the Mycobacterium leprae Genome Project: Structure and Function of the Rif-Str Regions, Mol. Microbiol., 7(2):207-214(1993)) and M. smegmatis (see: Levin, M. E. and Hatfull, G. F., Mycobacterium smegmatis RNA Polymerase: DNA Supercoiling, Action of Rifampin and Mechanism of Rifampin Resistance, Mol. Microbiol., 8(2):277-285(1993)).

Also, nucleotide sequence in a region of a rpoB gene is highly conserved in some mycobacteria other than Mycobacterium tuberculosis (see: Hunt, J. M., G. D. Roberts, L. Stockman, T. A. Felmiee and D. H. Persing, Detection of a Genetic Locus Encoding Resistance to Rifampin in Mycobacterial Cultures and in Clinical Specimens, Diagn. Microbiol. Infect. Dis., 18:219-272(1994); Whelen, A. C., T. A. Felmlee, J. M. Hunt, D. L. Williams, G. D. Roberts, L. Stockman and D. H. Persing, Direct Genotypic Detection of Mycobacterium tuberculosis Rifampin Resistance in Clinical Specimens by Using Single-tube Heminested PCR, J. Clin. Microbiol., 33:556-561(1995)).

In addition, a rpoB gene is used for phylogenetic establishment of Archaebacteria(see: Puhler, G., H. Leffers, F. Gropp, P. Palm, H. P. Klenk, F. Lottspeich, R. A. Garrett and W. Zillig, Archaebacterial DNA-dependent RNA Polymerases Testify to the Evolution of the Eukaryotic Nuclear Genome, Proc. Natl. Acad. Sci., U.S.A., 86:4569-4573(1989); Iwabe N., K. Kuma, H. Kishino, M. Hasegawa, and Miyata, Evolution of RNA Polymerases and Branching Patterns of the Three Major Groups of Archaebacteria, J. Mol. Evol., 32:70-78(1991); Klenk, H. P. and W. Zillg, DNA-dependent RNA Polymerase Subunit B as a Tool for Phylogenetic Reconstructions: Branching Topology of the Archaeal Domain, J. Mol. Evol., 38:420-432(1994); Zillig, W., H. P. Klenk, P. Palm, G. Puhler, F. Gropp, R. A. Garrett and H. Leffers, The Phylogenetic Relations of DNA-dependent RNA Polymerases of Archaebacteria, Eukaryotes, and Eubacteria, Can. J. Microbiol., 35:73-80(1989)), Eubacteria other than Staphylococcus aureus (see: Rowland G. C., M. Aboshkiwa and G. Coleman, Comparative Sequence Analysis and Predicted Phylogeny of the DNA-dependent RNA Polymerase Beta Subunits of Staphylococcus aureus and other Eubacteria, Biochem. Soc. Trans., 21:40S(1993)) and Plasmodium (see: Gardner, M. J., N. Goldman, P. Barnett, P. W. Moore, K. Rangachari, M. Strath, A. Whyte, D. H. Williamson and R. J. Wilson, Phylogenetic Analysis of the rpoB Gene from the Plastid-like DNA of Plasmodium falciparum, Mol. Biochem. Parasitor., 66:221-231(1994)).

SUMMARY OF THE INVENTION

The present inventors investigated whether a rpoB gene can be used as a criterion for mycobacterial phylogenetic analysis: That is, 342 bp of rpoB gene fragments, which corresponds to an amino acid sequence from 447th amino acid to 561st amino acid in E. coli, were amplified from 44 mycobacterial species, and 306 bp of nucleotide sequences except for primer sequences were determined and compared with each other. As a result, it was found that: rpoB sequences from the 44 mycobacterial species provide a basis for systematic phylogenetic relationship that can be used to identify clinically isolated mycobacteria which are pathogenic or potentially so; and, therefore, PCR-mediated sequence analysis of rpoB DNA can be regarded as a reliable and rapid method for the diagnosis of mycobacterial infection.

A primary object of the invention is, therefore, to provide a pair of PCR primers which specifically amplify rpoB gene fragments of bacterial species belonging to the genus Mycobacterium.

The other object of the invention is to provide species-specific nucleotide sequences of the rpoB gene fragments amplified by using the said PCR primers.

Another object of the invention is to provide a method for detecting and identifying various mycobacterial species by PCR-mediated sequencing of the rpoB gene fragments amplified from clinical isolates and determining phylogenetic relationship to the reference species.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:

FIGS. 1A-G show nucleotide sequences of the rpoB gene fragments(306 bp) of 44 mycobacterial species and 3 phylogenetically related non-mycobacterial species (SEQ ID NO:1 to SEQ ID NO:47), which were amplified by employing genus Mycobacterium-specific PCR primers.

FIGS. 2A-B show a phylogenetic tree based on the nucleotide sequences of the rpoB gene fragments (306 bp) of the 44 mycobacterial species and non-mycobacterial species.

FIGS. 3A-B show results of identification of clinical isolates by determining the phylogenetic relationship.

DETAILED DESCRIPTION OF THE INVENTION

Based on the previous reports that a nucleotide sequence in a region of a rpoB gene is highly conserved in mycobacteria, the present inventors first designed a pair of primers(5′-CGACCACTTCGGCAACCG-3′ (SEQ ID NO:48) and 5′-TCGATCGGGCACATCCGG-3′ (SEQ ID NO:49)) by which rpoB DNAs of bacteria belonging to genus Mycobacterium can be specifically amplified. Then, amplification of 342 bp of rpoB gene fragments from 44 mycobacterial species including M. tuberculosis were followed by employing polymerase chain reaction (PCR). In this connection, 342 bp of rpoB gene fragments from 3 kinds of non-mycobacteria including Corynebacterium diphtheriae, Nocardia nova and Rhodococcus equi which are phylogenetically related to mycobacteria were also amplified by using the said primers for further use as reference microorganisms to construct a phylogenetic tree. In addition, Staphylococcus aureus, Bacillus subtilis and E. coli were used as general bacteria for PCR. And then, the nucleotide sequences of the amplified rpoB gene fragments (306 bp) except the said primer sequences were determined, which revealed that there are species-specific nucleotide sequences which are not appeared in non-mycobacteria.

A phylogenetic tree was constructed, based on the total 50 rpoB nucleotide sequences which includes 44 mycobacterial species, 3 kinds of non-mycobacteria which are phylogenetically related to mycobacteria and 3 kinds of general bacteria. The phylogenetic tree provided an alternative for the conventional ambiguous mycobacterial systematics and, revealed that the comparative sequence analysis of the rpoB gene fragments (342 bp) can be used for characterization of mycobacteria. That is, slow- and rapid-growing mycobacteria were clearly identified, and each mycobacterial species was characterized as a distinct entity in the phylogenetic tree. In particular, pathogenic M. kansasii were easily distinguished from nonpathogenic M. gastri, although they have not been identified by a conventional 16S rRNA sequences. Members of MAC(or MAIS complex) such as M. avium, M. intracellulare and M. scrofulaceum, which have been regarded as phylogenetically related to the other were also clearly identified. By inferring the phylogenetic tree on the basis of reference species and using the rpoB sequences thus determined, clinical isolates could easily be identified.

Further, M. leprae, not yet cultivated in vitro was successfully identified by the procedure punch biopsy-PCR-rpoB sequence analysis.

Accordingly, the amplification of rpoB DNA followed by automated sequencing and the analysis of phylogenetic relationships can be used efficiently to detect and identify clinical isolates of mycobacteria which have not been identified by the conventional methods. In particular, this approach is useful for slowly growing, fastidious or uncultivable mycobacteria. Furthermore, in the case of M. tuberculosis, rifampin susceptibility can be simultaneously determined.

The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

EXAMPLE 1 Isolation of DNA from Various Mycobacterial Strains

M. leprae (Thai 53 strain) was prepared from the footpad of nude mice (nu/nu in Balb/c, BTK, U. K.), which was inoculated and maintained for 18 months. A punch biopsy specimen was obtained from an active lesion of a patient diagnosed on the basis of histological findings, AFB staining, and amplification of DNA encoding 18 kDa protein (see: Williams, D. L., T. P. Gillis, R. J. Booth, D. Looker and J. D. Watson, The Use of a Specific DNA Probe and Polymerase Chain Reaction for the Detection of Mycobacterium leprae, J. Infect. Dis., 162:193-200(1990)). The resected swollen footpads and biopsy specimen were homogenized in 2 ml phosphate buffered saline (PBS) using Mickle homogenizer (Mickle Laboratory Engineering, Surrey, U.K.). Supernatant was collected after settling down of tissue debris (1×g for 5 min) and M. leprae DNA was prepared by the aid of freezing-thawing technique. Other mycobacterial genomic DNAs were prepared by the Bead-beat/Phenol extraction method. A loopful culture of each mycobacteria was suspended with 200 μl of TEN buffer(10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl) placed in a 2.0 ml screw-cap microcentrifuge tube filled with 100 μl (packed volume) of beads (glass bead, diameter 0.1 mm; Biospec Products, Bartlesville, Okla., U.S.A.) and 100 μl phenol:chloroform:isopropylalcohol solution (50:49:1, v/v/v). To disrupt the bacteria, the tube was oscillated on a Mini-Bead beater (Biospec Products, Bartlesville, Okla., U.S.A.) for one minute, and to separate phases, the tube was centrifuged (12,000×g, 5 min). After the aqueous phase was transferred into another clean tube, 10 μl 3M sodium acetate and 250 μl ice-cold ethanol were added and, the mixture was kept at −20° C. for 10 minutes to precipitate the DNA. The DNA pellet was washed with 70% ethanol, solubilized with 60 μl TE buffer(10 mM Tris-HCl, 1 mM EDTA) and used as a template DNA for PCR for nucleotide sequencing in the following Examples. In this connection, the mycobacterial strains used for the extraction of genomic DNA are shown in Table 1 below, which were provided by American Type Culture Collection (ATCC), the Korean Institute of Tuberculosis, the Korean National Tuberculosis Association (KNTA), etc. 3 kinds of non-mycobacteria including Corynebacterium diphtheriae, Nocardia nova and Rhodococcus equi which are phylogenetically related to mycobacteria were used as references to construct a phylogenetic tree (see: Table 1), and Staphylococcus aureus, Bacillus subtilis and E. coli were used as general bacteria for PCR.

TABLE 1 44 Kinds of Mycobacteria and 3 Kinds of Non- mycobacteria Used for Comparative Sequence Analysis of rpoB Gene Species Strain Source* M. abscessus CAP97E-03 CPSNU M. africanum ATCC 25420 ATCC M. asiaticum ATCC 25276 ATCC M. aurum ATCC 23366 ATCC M. avium ATCC 25291 ATCC M. bovis ATCC 19210 ATCC M. bovis BCG French strain1173P2 KNTA M. celatum Type 1 ATCC 51131 ATCC M. celatum Type 2 ATCC 51130 ATCC M. chelonae ATCC 35749 ATCC M. chitae ATCC 19627 ATCC M. fallax ATCC 35219 ATCC M. flavescense ATCC 14474 ATCC M. fortuitum ATCC 6841 ATCC M. fortuitum 49403 ATCC 49403 ATCC M. gastri ATCC 15754 ATCC M. genavense ATCC 51233 ATCC M. gordonae ATCC 14470 ATCC M. haemophilum ATCC 29548 ATCC M. interjectum ATCC 51457 ATCC M. intermedium ATCC 51848 ATCC M. intracellulare ATCC 13950 ATCC M. kansasii ATCC 12478 ATCC M. leprae Thai 53 strain ICD M. malmoense ATCC 29571 ATCC M. marinum ATCC 927 ATCC M. neoaurum ATCC 25795 ATCC M. nonchromogenicum ATCC 19530 ATCC M. paratuberculosis ATCC 19698 ATCC M. phlei ATCC 11758 ATCC M. peregrinum ATCC 14467 ATCC M. scrofulaceum ATCC 19981 ATCC M. senegalense ATCC 35796 ATCC M. shimoidei ATCC 27962 ATCC M. simiae ATCC 25275 ATCC M. smegmatis ATCC 19420 ATCC M. szulgai ATCC 35799 ATCC M. terrae ATCC 15755 ATCC M. thermoresistibile ATCC 19527 ATCC M. triviale ATCC 23292 ATCC M. tuberculosis H37Rv ATCC 27294 ATCC M. ulcerans ATCC 19423 ATCC M. vaccae ATCC 15483 ATCC M. xenopi ATCC 19250 ATCC R. equi ATCC 10146 ATCC N. nova ATCC 21197 ATCC C. diphtheriae SNUMCd IMSNU *KNTA: The Korean Institute of Tuberculosis, Korean National Tuberculosis Association ICD: The Institute of Chronic Diseases, College of Medicines, Catholic University CPSNU: Department of Clinical Pathology, College of Medicines, Seoul National University IMSNU: Culture Collection Center, Institute of Microbiology, Seoul National University

EXAMPLE 2 PCR Amplification of rpoB Gene Fragments

Based on the report that a nucleotide sequence in a region of a rpoB gene is highly conserved in mycobacteria, a pair of primers specific to the genus Mycobacterium were designed as followings:

MF primer: 5′-CGACCACTTCGGCAACCG-3′ (SEQ ID NO:48)

MR primer: 5′-TCGATCGGGCACATCCGG-3′ (SEQ ID NO:49)

And then, 50 ng of genomic DNA of each bacterium isolated in Example 1 and 20 pmole of the said primers were added to a PCR mixture tube (Pre-mix Top, Bioneer, Korea), and distilled water was added in a final volume of 20 μl, and PCR was performed. In this connection, denaturation(95° C., 30 seconds), annealing (60° C., 30 seconds) and extension (72° C., 45 seconds) were carried out for 30 cycles, respectively, using a thermocycler (Model 9600, Perkin-Elmer Cetus, USA).

The PCR products thus amplified were electrophoresed on 3% agarose gel. As a result, it was found that the rpoB genes of only mycobacteria can be amplified by using the said primers, since the rpoB DNA fragments of 342 bp were amplified from 44 mycobacterial species and closely related species such as Rhodococcus, Nocardia and Corynebacterium, while no amplifications were observed from Staphylococcus, Streptococcus, Haemophillus, and enteric bacteria which can usually be isolated from the human body.

EXAMPLE 3 Determination of Nucleotide Sequences of rpoB Gene Fragments

The PCR products(DNAs) amplified in Example 2 were separated by 3% agarose gel electrophoresis, and purified using Qiaex II Gel Extraction Kit (QUIAGEN, Hilden, Germany). Nucleotide sequences (306 bp) of the purified rpoB DNAs except for the two primer sequences were determined by employing an automatic sequencer (ABI, USA). Also, rpoB DNA fragments of 3 kinds of controls shown in Example 1 were also amplified, and their nucleotide sequences were determined in the same manner.

For the sequencing reaction, 60 ng of template DNA, 3.2 pmole of each genus-specific primer and distilled water were mixed in a final volume of 10.5 μl. Then, 4 μl of 5×TACS buffer, 1 μl of dNTP mixture, 4 μl of termination-mixture, and 0.5 μl (5U/μl ) of Taq polymerase were added, and the reaction was carried out using 5% (v/v) dimethylsulfoxide for 30 cycles of 15 seconds at 95° C., 10 seconds at 50° C., and 4 minutes at 60° C. Both strands were sequenced for cross-check. FIGS. 1A-G show the nucleotide sequences (306 bp) of the amplified rpoB fragments of the 44 mycobacterial strains and the 3 non-mycobacterial strains. The (G+C) ratio of these amplified DNAs were 63-69%, which reflects the general phenotype of genus Mycobacterium (62-70%).

The nucleotide sequences thus determined were compared with one another for the investigation of pairwise similarity, which revealed that mycobacterial species are closely related with each other and are clearly distinguished from other bacterial genuses. In general, 85-100% similarity was observed among mycobacterial species, and there were no insertion or deletion. Interestingly, members of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. bovis BCG and M. africanum) were identical, which may be an evidence to support a hypothesis that M. bovis is a subspecies of M. tuberculosis as M. avium and M. paratuberculosis are (99.3<percent similarity).

Amino acid sequences deduced from the amplified rpoB DNA demonstrated 101 amino acid residues. As expected, amino acid sequences among mycobacterial species were also highly conserved, while variations were observed at 6 amino acids (458^(th), 468^(th), 531^(st), 539^(th), 541^(st) and 552^(nd)). Interestingly, instead of the M₄₆₈(ATG), which was found uniformly in most of the slow-growing mycobacteria, rapid-growing mycobacteria and M. terrae complex had L₄₆₈ (CTG, TTG or CTC) as non-mycobacteria. Among the investigated mycobacteria, only M. celatum which has been known to be rifampin-resistant had N₅₃₁ (AAC). That is one of the most frequent site of mutation representing rifampin susceptibility in M. tuberculosis[S531→L531 (TCG-TTG)].

EXAMPLE 4 Construction of a Phyogenetic Tree of Mycobacteria

A phylogenetic tree was constructed employing MegAlign package (Windows Version 3.12e, DNASTAR, Madison, Wis., USA) based on the following nucleotide sequences, in accordance with neighborhood joining method (see: FIGS. 2A-B):

1) rpoB DNA fragments (306 bp) of 44 kinds of mycobacteria;

2) the nucleotide sequences of rpoB of Gram-negative E. coli (V00340; ECOLI*), Gram-positive B. subtilis (L24376; BSUBT*), and Gram-positive S. aureus (X64172; SAURE*) registered in Gene Bank; and,

3) the nucleotide sequences of rpoB of C. diphtheriae, N. nova and R. equi.

As can be seen in FIGS. 2A-B, all of tested species showed good identification. An interesting feature is the natural coherence of the classical taxonomic distinction between rapid- and slow-growing species. Rapid-growing species are united to exclude the slow-growing lines. In this phylogenetic tree clustering of pathogenic and potentially pathogenic species is another characteristic. M. fortuitum, M. chelonae, and M. abscessus, which are included in the taxonomic groups of pathogenic, rapidly growing mycobacteria, form a distinct cluster. M. haemophilum is the species closest to M. leprae as the result from 23S rRNA sequence analysis (see: Stone, B. B., R. M. Nietupski, G. L. Breton and W. G. Weisberg, Comparison of Mycobacterium 23S rRNA Sequences by High-temperature Reverse Transcription and PCR, Int. J. Syst. Bacteriol., 45:811-819, 1995).

In addition, characteristic findings were observed among the debated and newly recognized species. For example, M. kansasli and M. gastri are clearly identified in terms of taxonomy, though they were indistinguishable from each other by a conventional 16S rRNA sequence analysis. M. intracellulare, which has been long regarded to be closely related to M. avium, is close to M. asiaticum, whereas M. avium is clustered with M. paratuberculosis, M. celatum and M. scrofulaceum. On the other hand, M. celatum is distinctly separated from M. avium, M. paratuberculosis and M. scrofulaceum, which suggests that it can reasonably be regarded as a distinct species.

EXAMPLE 5 Identification of Clinically Isolated Mycobacterial Species (wild type) from Patients

In order to investigate whether comparative rpoB DNA sequencing can be really applied to wild type mycobacteria, i.e., clinically isolated species from patients, 7 mycobacterial species which were isolated from patients and had already been identified biochemically, were employed. Amplification and PCR-mediated sequencing of rpoB gene fragments were accomplished according to the methods disclosed in Examples 2 and 3, respectively. And then, phylogenetic relationship was determined by inferring a phylogenetic tree together with the reference species disclosed in Table 1 (see: FIGS. 3A-B). In FIGS. 3A-B, Lep-clin., Tub-clin., Kan(2-1).seq, MAC-clin.1, MAC-clin.2, For-clin.  and Abs-clin. represent the 7 clinical isolates of microbacteria, respectively. As a result, M. leprae, M. tuberculosis, MAC, M. kansasii, M. fortuitum, and M. abscessus which had been confirmed in the conventional way, were successfully identified by forming a tight cluster with known reference species. Only two nucleotides were changed in the clinical isolate of M. leprae [T₄₉₁ (ACG→ACC), L₅₃₈ (TTG→CTG)]. Others showed one nucleotide difference or identical rpoB sequences with the type strains. Further, the clinical isolate of M. tuberculosis, i.e., Tub-clin. was found to have a missense mutation of S₅₃₁→L₅₃₁(TCG→TTG) and rifampin resitance.

These above results illustrate that: rpoB sequences from 44 mycobacterial species provide a basis for systematic phylogenetic relationship that can be used to identify clinically isolated mycobacteria that are pathogenic or potentially so; and, therefore, PCR-mediated sequence analysis of rpoB DNA can be regarded as a reliable and rapid method for the diagnosis of mycobacterial infection.

As clearly illustrated and demonstrated as aboves, the present invention provides a method for diagnosis of mycobacterial infection by comparative sequence analysis of rpoB gene coding for β-subunit of RNA polymerase. In accordance with the present invention, it was found that: rpoB sequences from 44 mycobacterial species provide a basis for systematic phylogenetic relationship which can be used to identify clinically isolated mycobacteria that are pathogenic or potentially so. Accordingly, the amplification of rpoB DNA followed by automated sequencing and the analysis of phylogenetic relationships with the reference species can be used efficiently to detect and identify clinical isolates of mycobacteria which have not been identified by the conventional methods. In particular, this approach is useful for slowly growing, fastidious or uncultivable mycobacteria. Furthermore, in the case of M. tuberculosis, rifampin susceptibility can be simultaneously determined. Thus, the PCR-mediated comparative sequence analysis of rpoB DNA of the invention can be regarded as a reliable and rapid method for the diagnosis of mycobacterial infection.

50 1 306 DNA Mycobacterium abscessus 1 tgcgtaccgt cggcgagctg attcagaacc agatccgggt cggcctgtcc cgtatggagc 60 gcgtcgtgcg tgagcgcatg accacgcagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggaaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg tggtctgacc cgtgaccgcg ccggcctcga ggtccgcgac gtgcacccct 300 cgcact 306 2 306 DNA Mycobacterium africanum 2 tgcgtacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtggaggc gatcacaccg cagacgttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 aattcatgga ccagaacaac ccgctgtcgg ggttgaccca caagcgccga ctgtcggcgc 240 tggggcccgg cggtctgtca cgtgagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 3 306 DNA Mycobacterium asiaticum 3 tgcgcaccgt gggcgagttg atccagaacc agatccgggt cggcatgtcc cggatggagc 60 gcgtcgtccg cgagcggatg accactcagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg gccggtcgtt gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctttcgg gtctgaccca caagcgccgc ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgtg ccgggctgga agtgcgtgac gtgcacccct 300 cgcact 306 4 306 DNA Mycobacterium aurum 4 tgcgtaccgt cggcgagctg atccagaacc agatccgcgt cggcctctcg cgtatggagc 60 gtgtcgtgcg tgagcgcatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcacgtcg cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgccgc ctgtcggcgc 240 tgggcccggg tggtctgtcc cgtgagcgcg ccggcctcga ggtccgcgac gtgcactcca 300 gccact 306 5 306 DNA Mycobacterium avium 5 tgcgcaccgt cggtgagctg atccagaacc agatccgggt cggcatgtcc cggatggagc 60 gcgtcgtccg cgagcggatg accacccagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgtccc 180 agttcatgga ccagaacaac ccgctgtcgg ggctcaccca caagcgccgc ctgtcggcgc 240 tgggcccggg tggtctgtcc cgggagcggg ccgggctgga ggtccgcgac gtgcacccgt 300 cccact 306 6 306 DNA Mycobacterium bovis 6 tgcgtacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtggaggc gatcacaccg cagacgttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 aattcatgga ccagaacaac ccgctgtcgg ggttgaccca caagcgccga ctgtcggcgc 240 tggggcccgg cggtctgtca ccgtgacgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 7 306 DNA Mycobacterium bovis BCG 7 tgcgtacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtggaggc gatcacaccg cagacgttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 aattcatgga ccagaacaac ccgctgtcgg ggttgaccca caagcgccga ctgtcggcgc 240 tggggcccgg cggtctgtca cgtgagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 8 306 DNA Mycobacterium celatum Type1 8 ttcgtaccgt cggtgagctg atccagaacc agatccgagt cggcatgtcc cgcatggagc 60 gggtggtccg cgagcggatg accactcagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtccg ggctgaccca caagcggcgc ctgaacgcac 240 tgggcccggg tggtctgtcc cgggagcggg cgggcctcga ggtgcgcgac gtgcacccga 300 gtcact 306 9 306 DNA Mycobacterium celatum Type2 9 ttcgtaccgt cggcgagctg atccagaacc agatccgggt cggtatgtcg aggatggagc 60 gggtggtccg cgagcggatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tccggtcgtg gcggccatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccattgtccg ggctgaccca caagcgtcgc ctgaacgcgc 240 tcggcccggg tggtctgtcc cgggagcggg ccggcctgga ggtccgcgac gtgcacccga 300 gccact 306 10 306 DNA Mycobacterium chelonae 10 tgcgtaccgt cggcgagctg atccagaacc agatccgggt cggcctgtcg cgtatggagc 60 gcgtcgtgcg tgagcgcatg accactcagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggaaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctttcgg gtctgaccca caagcgtcgt ctgtcggctc 240 tgggccccgg tggtctgacc cgtgaccgcg ctggccttga ggtccgcgac gtgcacccct 300 cgcact 306 11 306 DNA Mycobacterium chitae 11 tgcgcaccgt gggtgagctg atccagaacc agatccgggt cggcctgtcc cgcatggagc 60 gcgtcgtgcg cgagcggatg accacccagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtccg ggctgaccca caagcgtcgt ctctcggcgc 240 tcgggcccgg cggtctgtcc cgtgagcgcg ccggtctcga ggttcgtgac gtgcacccgt 300 cgcact 306 12 306 DNA Mycobacterium fallax 12 tgcgcaccgt gggcgagctg atccagaacc agatccgggt cggcctgtcc cggatggagc 60 gcgtcgtccg cgagcggatg accacccagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtggtg gcggcgatca aggagttctt cgggaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctgaccca caagcgccgg ctgtccgcgc 240 ttggccccgg cggtctgtcc cgtgagcgcg ccggcctgga ggtccgcgac gtgcacgcca 300 gccact 306 13 306 DNA Mycobacterium flavescens 13 tgcgcaccgt cggcgagctg atccagaacc agatccgggt cggcctgtcg cggatggagc 60 gcgtcgtccg tgagcggatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggtacgtcg cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgccgc ctgtcggcgc 240 tgggccccgg tggtctgtcc cgtgagcgcg ccggcctcga agtccgtgac gtgcacccgt 300 cgcact 306 14 306 DNA Mycobacterium fortuitum 14 tgcgcaccgt gggcgagctg atccagaacc agatccgcgt cggcctgtcc cgcatggagc 60 gcgtcgtgcg tgagcgcatg accacccagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggaacgtcg cagctgtcgc 180 agttcatgga tcagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggccttga ggtccgcgac gtccactcgt 300 cgcact 306 15 306 DNA Mycobacterium fortuitum 49403 15 tgcgcaccgt gggcgagctg atccagaacc agatccgggt cggcctgtcc cgcatggagc 60 gcgtcgtgcg tgagcgcatg accacccagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggaacgtcg cagctgtcgc 180 agttcatgga tcagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggccttga ggtccgcgac gtccactcgt 300 cgcact 306 16 306 DNA Mycobacterium gastri 16 tgcgcacggt gggcgagctg atccagaacc agatccgggt cggcatgtcc aggatggagc 60 gcgtcgtccg ggagcggatg accactcagg acgtcgaggc catcacgccg cagacgctga 120 tcaacattcg cccggtggtc gctgccatta aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctgaccca caagcgccgg ctttcggcgc 240 tgggccccgg cggtctgtca cgtgagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 17 306 DNA Mycobacterium genavense 17 tgcgcacggt gggcgatctg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg tgagcggatg accactcagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg tccggttgtg gcggcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcag gtctcaccca caagcgccgg ttgtcggcgc 240 tggggccggg cggtctgtcc cgtgagcggg cgggcctcga ggtccgcgac gtgcacccgt 300 ctcact 306 18 306 DNA Mycobacterium gordonae 18 tgcgcaccgt gggcgagctg atccagaacc agatccgggt cggcatgtcc cggatggagc 60 gcgtcgtgcg cgaccggatg accactcagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg gccggtcgtc gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgc 240 tggggccggg tggtctgtcc cgtgagcgtg cgggtctgga agtacgtgac gtgcacccgt 300 cgcact 306 19 306 DNA Mycobacterium haemophilum 19 tgcgcacggt cggcgaattg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accactcagg acgtcgaggc gatcacgccg cagacgctga 120 tcaatatccg gccggtggtg gccgcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtccg gcctaaccca caagcgccgg ctgtcggcgc 240 tggggccggg cggtctgtcg cgtgagcgtg ccgggctaga ggtccgcgac gtgcacccgt 300 cgcact 306 20 306 DNA Mycobacterium interjectum 20 tgcgtaccgt cggcgagctg atccagaacc agatccgggt cggcatgtcc cgcatggagc 60 gcgttgtccg cgagcggatg accactcagg acgtcgaggc catcacgccg cagaccttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgt 240 tgggcccggg tggtctgtcg cgtgagcgtg ccgggctgga agtccgtgac gtgcacccgt 300 cgcact 306 21 306 DNA Mycobacterium intermedium 21 tgcgcaccgt cggtgagctg atccagaacc agatccgggt cggcatgtcc aggatggagc 60 gcgtcgtccg ggagcggatg accacccagg acgtcgaggc gatcacgccg cagacgctga 120 tcaacatccg gccggtcgtc gccgcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctcaccca caagcgccgc ctgtcggcgc 240 tgggcccggg cggtctgtcc cgcgagcggg ccggcctcga ggtccgcgac gtgcacccga 300 accact 306 22 306 DNA Mycobacterium intracellulare 22 tgcgcaccgt gggtgagctg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gcgtcgtccg cgagcggatg accacgcagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg gccggtcgtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 agttcatgga ccagaacaac ccgctgtccg gtctgaccca caagcgccgc ctctcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggcctgga ggtccgtgac gtccacccct 300 cgcact 306 23 306 DNA Mycobacterium kansasii 23 tgcgtaccgt cggcgagctg atccagaacc agatccgggt cggcatgtcg aggatggagc 60 gggtggtccg ggaacggatg accactcagg acgtcgaggc gatcacgccg cagacactga 120 tcaacatccg cccggtggtc gccgccatca aggagttctt cggcaccagc cagctctccc 180 agttcatgga ccagaacaac ccgctgtcgg gcctcaccca caagcgccgg ctttcggcgc 240 tggggccggg cggtctgtcc cgggagcgtg ccgggctgga agtgcgtgac gtgcacccgt 300 cgcact 306 24 306 DNA Mycobacterium leprae 24 tgcgcacggt cggcgaattg atccagaacc agatccgggt cggtatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacgatca cgccgcagac gctgatcaat atccgtccgg 120 tggtcgccgc tatcaaggaa ttcttcggca ccagccagct gtcgcagttc atggatcaga 180 acaaccctct gtcgggcctg acccacaagc gccggctgtc ggcgctgggc ccgggtggtt 240 tgtcgcgtga gcgtgccggg ctagaggtcc gtgacgtgca cccttcgcac tccaggacgt 300 cgaggc 306 25 306 DNA Mycobacterium malmoense 25 tgcgcacggt cggggagctg atccagaacc agatccgcgt cggcatgtcg cggatggagc 60 gcgtcgtccg ggagcggatg accacccagg acgtcgaggc gatcacgccg cagacgctga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg ggctgaccca caagcgccgg ctgtcggcgc 240 tgggcccggg tggtctgtcg cgtgagcgtg ccggcttgga ggtccgtgac gtgcacccgt 300 cgcact 306 26 306 DNA Mycobacterium marinum 26 tgcgcacggt gggtgagctg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtcgaggc gatcacgccg cagacgctga 120 tcaacatccg tccggtcgtt gcggcgatca aggagttctt cggaaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctctccg gtctcaccca caagcgccgc ctctcggcgc 240 tggggccggg cggtctgtcc cgtgagcgcg ccggtctgga agttcgtgac gtgcacccgt 300 cgcact 306 27 306 DNA Mycobacterium neoaurum 27 tgcgcaccgt gggtgagctg atccagaatc agatccgggt cggcctgtcg cgcatggagc 60 gggtcgtgcg cgagcgcatg accacccagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cgggaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg tggtctgtcc cgtgagcgtg ccggacttga ggtccgcgac gtgcactcca 300 gccact 306 28 306 DNA Mycobacterium nonchromogenicum 28 tgcgcaccgt gggtgagctg atccagaacc agatccgggt cgggctgtcc cggatggagc 60 gcgtggtccg cgagcggatg accacccagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg cccggtggtc gccgccatca aggaattctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcag gtctgaccca caagcggcgt ctgtcggcgc 240 tgggccccgg tggtctgtcg cgtgagcgcg ccggcctgga agttcgtgac gtgcacccgt 300 cccact 306 29 306 DNA Mycobacterium paratuberculosis 29 tgcgcaccgt cggtgagctg atccagaacc agatccgggt cggcatgtcc cggatggagc 60 gcgtcgtccg cgagcggatg accacccagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagttgtccc 180 agttcatgga ccagaacaac ccgctgtcgg ggctcaccca caagcgccgc ctgtcggcgc 240 tgggcccggg tggtctgtcc cgggagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cccact 306 30 306 DNA Mycobacterium peregrinum 30 tgcgcaccgt cggtgagctg atccagaacc agatccgggt cggcctgtcg cgtatggagc 60 gtgtcgtgcg tgagcgcatg accacccagg acgtcgaggc gatcaccccg cagaccctga 120 tcaacatccg ccccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggccttga ggtccgcgac gtgcactcca 300 gccact 306 31 306 DNA Mycobacterium phlei 31 tgcgcaccgt cggcgagctg atccagaacc agatccgggt cggcctgtcg cgtatggagc 60 gcgtcgtgcg cgagcgcatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgccgc ctgtcggcgc 240 tgggcccggg cggtctgtcc cgtgagcgcg ccggcctcga ggtccgcgac gtgcaccaca 300 gccact 306 32 306 DNA Mycobacterium scrofulaceum 32 tgcgcaccgt cggggagctg atccagaacc agatccgggt cggcatgtcc cgcatggagc 60 gggtcgtccg cgagcggatg accacgcagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg gccggtcgtg gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctgaccca caagcgccgc ctgtcggcgc 240 tgggcccggg tggtctgtcc cgcgagcggg ccgggctgga ggtccgggac gtgcacccgt 300 cgcact 306 33 306 DNA Mycobacterium senegalense 33 tgcgcaccgt gggtgagctg atccagaacc agatccgggt cggcctgtcc cgcatggagc 60 gcgtcgtgcg tgagcggatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggtaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctttcgg gtctgaccca caagcgtcgc ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgtg ccggccttga ggtccgcgac gtgcacgcca 300 gccact 306 34 306 DNA Mycobacterium shimoidei 34 tgcgcacggt gggtgagctg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtcgaggc gatcacgccg cagacgctga 120 tcaacatccg tccggtcgtt gccgcgatca aggagttctt cggaaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtccg gtctcaccca caagcgccgc ctctcggcgc 240 tggggccggg cggtctgtcc cgtgagcgtg ccgggctgga agttcgtgac gtgcacccgt 300 cgcact 306 35 306 DNA Mycobacterium simiae 35 tgcgcacggt gggcgaactg atccaaaacc agatccgcgt cggcatgtcg cgtatggagc 60 gtgtcgtccg tgagcggatg accactcagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg tccggttgtg gcggcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcag gtctcaccca caagcgccgg ttgtcggcgc 240 tggggccggg cggtctgtcc cgtgagcggg cgggcctcga ggtccgcgac gtgcacccgt 300 cgcact 306 36 306 DNA Mycobacterium smegmatis 36 tgcgcaccgt cggtgagctg atccagaacc agatccgcgt gggcctgtcc cgcatggagc 60 gtgtcgtgcg tgagcgcatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgt ctttcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ctggcctcga ggtccgcgac gtgcacccca 300 gccact 306 37 306 DNA Mycobacterium szulgai 37 tgcgcaccgt gggcgagttg attcagaacc agatccgggt cggcatgtcc cggatggagc 60 gcgtcgtgcg cgagcggatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg gcccgtcgtc gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctctccg gtctgacgca caagcggcgt ctgtccgctc 240 tggggccggg cggtctgtcc cgtgagcggg ccgggctgga ggtccgtgac gtgcacccgt 300 cgcact 306 38 306 DNA Mycobacterium terrae 38 tgcgcacggt gggtgagctg atccagaacc agatccgggt cgggttgtcc cggatggagc 60 gtgtggtccg cgagcggatg accacccagg acgtcgaggc catcacgccg cagaccctga 120 tcaacatccg cccggtggtc gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgccgg ctgtcggcgc 240 tgggcccggg tggtctgtcc cgtgagcgtg ccgggcttga ggtccgtgac gtgcacccgt 300 cccact 306 39 306 DNA Mycobacterium thermoresistibile 39 tgcgcaccgt cggcgagctg atccagaacc agatccgggt cggcctgtcc cgcatggagc 60 gcgtcgtgcg cgagcggatg accacccagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg ccccgtcgtg gcggcgatca aggagttctt cggcaccagc cagctgagcc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgccgg ctgtcggcgc 240 tgggcccggg cggtctgagc cgggagcgcg ccggcctcga ggtccgcgac gtccacccgt 300 cgcact 306 40 306 DNA Mycobacterium triviale 40 tgcgcaccgt cggggagttg atccagaacc agatccgggt cgggctgtcc cggatggagc 60 gggtggtgcg cgagcggatg accacccagg atgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg cccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtccg ggctgaccca caagcgccgg ctgtcggcgc 240 tggggcccgg cgggctctcc cgggagcggg ccgggctgga ggtccgcgac gtgcacccca 300 gccact 306 41 306 DNA Mycobacterium tuberculosis 41 tgcgtacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtggaggc gatcacaccg cagacgttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 aattcatgga ccagaacaac ccgctgtcgg ggttgaccca caagcgccga ctgtcggcgc 240 tggggcccgg cggtctgtca cgtgagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 42 306 DNA Mycobacterium ulcerans 42 tgcgcacggt gggtgagctg atccagaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg atgtcgaggc gatcacgccg cagacgctga 120 tcaacatccg tccggtcgtt gccgcgatca aggagttctt cggaaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctctccg gtctcaccca caagcgccgc ctctcggcgc 240 tggggccggg cggtctgtcc cgtgagcgcg ccggtctgga agttcgtgac gtgcacccgt 300 cgcact 306 43 306 DNA Mycobacterium vaccae 43 tgcgcacggt cggtgagctg atccagaacc agatccgcgt cggcctctcg cgtatggagc 60 gtgtcgtccg cgagcggatg accacccagg acgtcgaggc gatcactccg cagaccctga 120 tcaacatccg tcccgtcgtg gcggcgatca aggaattctt cggcaccagc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gtctgaccca caagcgtcgc ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggcctcga ggtccgcgac gtgcactcca 300 gccact 306 44 306 DNA Mycobacterium xenopi 44 tgcgcacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg aggatggagc 60 gggtggtccg cgagcggatg accactcagg acgtcgaggc gatcaccccg cagaccttga 120 tcaacatccg ccccgtggtg gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga tcagaacaac ccgctgtcgg ggctcaccca caagcggcgg ctctcggcgc 240 ttggtccggg cggtctgtcg cgcgagcggg ccgggctgga ggtccgtgac gtgcactcga 300 gccact 306 45 306 DNA Corynebacterium diphtheriae 45 tgcgtaccgt cggcgagctg atccaaaacc aggttcgtgt gggtctctcc cgcatggagc 60 gcgttgttcg cgagcgcatg accactcagg atgctgagtc gatcacccct acctcgctga 120 tcaacgttcg ccctgtttct gccgccatcc gcgagttctt cggaacctca cagctatcgc 180 agttcatgga ccagaacaac tctctgtccg gtctgaccca caagcgtcgt ctctccgcac 240 tgggcccagg tggcctgtcg cgtgagcgcg ccggcattga ggtccgagac gttcacgctt 300 ctcact 306 46 306 DNA Nocardia nova 46 tccgcacggt cggcgagttg atccagaacc agatccgcgt cggcctctcg cggatggagc 60 gggtggtccg ggaacggatg accacccagg acgtcgaggc catcactccg cagaccctga 120 tcaacatccg tccgatcacg gcggcgctcc gggagttctt cggcacctca cagctgtcgc 180 agttcatgga ccaaaacaac ccactgtcgg gtctgaccca caagcgtcga ctctcggcgc 240 tggggcccgg tggtctgtcc cgtgagcgcg ccggcctgga agtccgcgac gtgcacccct 300 cgcact 306 47 306 DNA Rhodococcus equi 47 tgcgcacggt gggcgagctg atccagaacc agatccgcgt gggcctgtcc cgcatggagc 60 gcgtcgtccg cgagcgcatg acgactcagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg cccggtcgtc gccgcgatca aggagttctt cggaacctcc cagctgtcgc 180 agttcatgga ccagaacaac ccgctgtcgg gcctgaccca caagcgtcgt ctgtcggcgc 240 tgggccccgg cggtctgtcc cgtgagcgcg ccggcctcga ggtgcgagac gtccacccgt 300 cgcact 306 48 18 DNA Artificial Sequence synthesized oligonucleotide 48 cgaccacttc ggcaaccg 18 49 18 DNA Artificial Sequence synthesized oligonucleotide 49 tcgatcgggc acatccgg 18 50 306 DNA Mycobacterium tuberculosis 50 tgcgtacggt cggcgagctg atccaaaacc agatccgggt cggcatgtcg cggatggagc 60 gggtggtccg ggagcggatg accacccagg acgtggaggc gatcacaccg cagacgttga 120 tcaacatccg gccggtggtc gccgcgatca aggagttctt cggcaccagc cagctgagcc 180 aattcatgga ccagaacaac ccgctgtcgg ggttgaccca caagcgccga ctgttggcgc 240 tggggcccgg cggtctgtca cgtgagcgtg ccgggctgga ggtccgcgac gtgcacccgt 300 cgcact 306 

What is claimed is:
 1. A pair of isolated PCR primers for sequence-specific amplification of rpoB gene of mycobacterial species whose nucleotide sequences consist of SEQ ID NO: 48 and SEQ ID NO:49.
 2. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium abscessus as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:1.
 3. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacteriurn africanum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:2.
 4. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium asiatictim as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:3.
 5. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium aurum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:4.
 6. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium avium as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:5.
 7. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium bovis as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:6.
 8. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium bovis BCG as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:7.
 9. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium celatum type I as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:8.
 10. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium celatunm type II as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:9.
 11. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium chelonae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:10.
 12. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium chitae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:11.
 13. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium fallax as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:12.
 14. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium flavescense as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:13.
 15. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium fortuitum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:14.
 16. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium fortuitum 49403 as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:15.
 17. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium gastri as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:16.
 18. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium genavense as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:17.
 19. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium gordonae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:18.
 20. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium haemophilum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:19.
 21. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium interjectum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:20.
 22. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium intermedium as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:21.
 23. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium intracellulare as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:22.
 24. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium kansasii as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consistino of SEQ ID NO:23.
 25. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium leprae (Thai 53) as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:24.
 26. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium malmoense as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:25.
 27. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium marinum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:26.
 28. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium neoaurum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:27.
 29. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium nonchromogenicum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:28.
 30. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium paratuberculosis as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:29.
 31. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium peregrinum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:30.
 32. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium phlei as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:31.
 33. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium scrofulaceum as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:32.
 34. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium senegalense as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:33.
 35. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium shimoidei as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:34.
 36. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium simiae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:35.
 37. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium smegmatis as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:36.
 38. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium szulgai as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:37.
 39. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium terrae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:38.
 40. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium thermoresistable as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:39.
 41. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium triviale as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:40.
 42. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacteriuim ulcerans as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:42.
 43. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium vaccae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:43.
 44. An rpoB gene fragment amplified by using isolated genomic DNA of Mycobacterium xenopi as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:44.
 45. An rpoB gene fragment amplified by using isolated genomic DNA of Corynebacterium diphtheriae as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:45.
 46. An rpoB gene fragment amplified by using isolated genomic DNA of Nocardia nova as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:46.
 47. An rpoB gene fragment amplified by using isolated genomic DNA of Rhodococcus equi as a template and both of the PCR primers of claim 1, the nucleotide sequence of said fragment consisting of SEQ ID NO:47.
 48. A method for detecting and identifying mycobacterial species, comprising the steps of: a) amplifying a 342 bp-long fragment of rpoB gene using both of the PCR primers of claim 1 and a template DNA purified from a clinically isolated mycobacterium; b) determining a complete DNA sequence of the amplified 342 bp rpoB gene fragment, with the exception of 18nt-long 5′ and 3′ sequences corresponding to both of said PCR primers, and c) inferring a phylogenetic relationship of said DNA sequence to the phylogenetic tree of FIG. 2, said tree having been prepared from the reference sequences shown in FIG. 1 with tlhe use of sequence analysis software.
 49. An rpoB gene fragment amplified by using both of the PCR primers of claim 1 and by using as a template genomic DNA isolated from a clinical strain of Mycobacterium tuberculosis having a missense mutation of TCG→TTG (S₅₃₁→L₅₃₁), the nucleotide sequence of said fragment consisting of SEQ ID NO:50. 